Epidemiological observations suggest that V. cholerae O1, that cause Asiatic cholera, exists as an indigenous member of the aquatic flora between outbreaks of human disease. Recently investigators in the Schoolnik lab have isolated a novel extracellular polysaccharide (EPS) from the rugose colonial variant of V. cholerae O1 El Tor. Chemical characterization of EPS shows that it is distinct from the O1 LPS and the O139 capsular polysaccharide and morphological studies suggest that it mediates biofilm formation by serving as an interbacterial matrix that causes bacteria to aggregate and attach to inanimate surfaces. Purified EPS also protects the smooth colonial variant of the same strain from the bactericidal effects of chlorine. Insertion mutagenesis of the rugose variant led to the identification of a heretofore unknown cluster of genes, designated vps, with sequences that share homology with polysaccharide biosynthetic genes from other species. Another class of mutants sustained insertions in genes homologous to two-component regulatory systems. These preliminary data have led to the hypothesis that EPS expression confers a survival advantage in environmental aquatic habitats. To test this hypothesis Dr. Schoolnik proposes to complete the structural characterization of EPS; characterize the vps gene cluster; prepare knockout mutants of selected vps genes and test these mutants for their capacity to survive in experimental microcosms that simulate natural aquatic habitats. Dr. Schoolnik's group will also identify physiologic factors that control EPS expression and study how these signals activate vps gene transcription. Finally, in collaborative studies conducted in Bangladesh, they will attempt to learn if V. cholerae O1 expresses EPS in naturally infected water sources. Taken together, these studies should shed some light on why this species continues to cause epidemics as a "re-emerging" infectious agent. The grant proposal is divided into three specific aims:(1) Continue to examine the structure and biosynthesis of EPS by completing the physical/chemical structural characterization, and by identifying the genes of the vps gene cluster by mutational analysis and complementation on clones. (2) Study the regulation of vps gene expression using vps gene fusions to ascertain vps gene expression under different growth conditions, including biofilm formation, murine intestinal growth, and algal co-culture. Identify regulatory genes that mediate this regulation by the isolation of mutants with altered regulation. (3) Study the function of EPS by determining the effects of vps mutants on survival under various conditions, and ascertaining the relative abundance of EPS and O1 LPS present on V. cholerae found in biofilm layers in Bangladesh. Determine if there is a direct correlation between the rugose morphotype and EPS production.